Evaluation and automation of hematopoietic chimerism analysis based on real-time quantitative polymerase chain reaction.

Abstract Chimerism analysis is an essential tool in the follow-up of patients after allogeneic stem cell transplantation. High-resolution methods for chimerism analysis based on real-time quantitative polymerase chain reaction (RQ-PCR) with a detection limit of 0.1% marker-specific cells are especially valuable in the detection of patient-derived subpopulations for the monitoring of minimal residual disease. Using artificial chimeric mixtures of genotypically different cells, we optimized and evaluated the intrasample variation, accuracy, and detection limit of chimerism analysis based on RQ-PCR of short insertion and deletion polymorphisms. Furthermore, automated setup by robot was evaluated. The results were accurate, with acceptable intrasample variation at and above 0.1% marker-specific cells. The sensitivity was mainly limited by background values. Chimerism results based on RQ-PCR were similar to results based on PCR of short tandem repeats when samples from recipients of transplants with nonmyeloablative conditioning were analyzed. Furthermore, automated setup was feasible in a time-, labor-, and reagent-conserving manner.     

IDO1 and IDO2 gene expression analysis by quantitative polymerase chain reaction

Immunomodulatory properties of IDO1 relate to tryptophan catabolism. The degradation of tryptophan by IDO1 leads to suppression of T cell responses. Recently, another enzyme with IDO-like activity, indoleamine 2,3-dioxygenase-like-protein 1 (INDOL1, IDO2), has been described in both mice and humans. In order to study the gene expression of IDO1 and IDO2, we have developed a quantitative PCR (qPCR) assay. In an exploratory application to the study of the differential expression of IDO1 and IDO2 by professional antigen-presenting cells and MSCs (mesenchymal stromal cells) under the influence of interferon-γ (IFN-γ) and T-lymphocyte conditioned media (TCM), substantial differences were observed. IDO expression measured by qPCR was valid and reliable in the cell types investigated. Further studies are needed to delineate factors driving IDO expression in MSCs.     

Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus isolation, and ELISAs for antigen and antibody) were used in parallel. Each inoculated pig developed a significant viraemia and clinical disease, and excreted virus, which was transmitted to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs.     

实时荧光定量PCR检测乳腺癌SOCS2基因表达

目的检测乳腺良恶性组织中细胞因子信号转导抑制因子（supressors of cytokine signaling,SOCS2）的mRNA表达及其临床病理意义。方法以18SrRNA为内j对照采用实时荧光定量PCR法,定量测定新鲜乳腺癌组织、癌旁组织和乳腺良性增生组织中。SOCS2 mRNA的表达并分析其与临床病理指标的关系。结果各组阳性率：乳腺癌35.00％（14／40）、癌旁乳腺组织100％（15／15）和乳腺良性增生组织81.25％（13／16）,其表达之间的差异具有统计学意义（P〈0．05）;乳腺癌组织的SOCS2表达水平与TNM分期和淋巴结转移呈明显的相关性（P〈0,05）。结论SOCS2在乳腺癌中的表达可能提示患者具有较好的预后。     

LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法的建立

目的 人副流感病毒1,2,3型是呼吸道感染的主要病原.本研究建立了特异、快速、灵敏的多重荧光定量RT-PCR方法用于人副流感1,2,3型病毒临床标本检测.方法 针对人副流感1,2,3型病毒设计特异性引物探针,优化荧光RT-PCR反应条件.应用体外转录方法分别制备人副流感1,2,3型病毒的标准品.验证荧光定量RT-PCR方法的特异性,敏感性和稳定性.结果 该方法对人副流感1,2,3型病毒核酸检测有高度特异性,检测的灵敏度HPIV1为10个拷贝,HPIV2为100个拷贝,HPIV3为100个拷贝.可从临床患者鼻咽吸出物标本中直接检出.结论 本研究建立的LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法具有较高的特异性和敏感性.适用于临床早期诊断和实验室病原谱筛查.